Objective: To use recombinant plasmid pcDNA expressing multiple antigen of glucosyltransferase-B (gtf-B) as gene vaccine, to investigate the immunization effects of different immunization routes. Methods: Eighteen Wistar rats were divided into three groups: quadriceps injection group, intranasal irrigation group and submandibular gland-targeted infection group. The serum IgG and salivary IgA were assayed by ELISA after pcDNA3-gtfB immunization. The serum IgG and salivary IgA in the three groups were compared by statistical one-way ANOVA. Results: Serum IgG of quadriceps injection was much higher than that of the other two groups (P < 0.01). Salivary IgA of submandibular gland-targeted injection was much higher than that of the other two groups (P < 0.01). Conclusion: It is indicated that pcDNA3-gtfB is a good candidate for anticarious gene vaccine; submandibular gland-targeted injection is an effective immunization route for stimulating salivary IgA.